Cellular reprogramming of induced cardiomyocytes (iCMs) holds promise as a novel therapy for the treatment of heart failure, a common and morbid disease that is caused by irreversible loss of functional cardiomyocytes (CMs). We and others have showed that in a murine model of acute myocardial infarction, delivery of three transcription factors, Gata4, Mef2c and Tbx5, converted endogenous cardiac fibroblasts into functional iCMs. These iCMs integrated electrically and mechanically with surrounding myocardium, resulting in a reduction in scar size and an improvement in heart function. These findings suggest that iCM reprogramming might be a promising novel approach to regenerate functional CMs in vivo for patients with heart disease. However, our limited understanding of the underlying molecular mechanisms of iCM reprogramming has significantly hindered its clinical applicability. In this proposal, we will take advantage of our expertise in iCM reprogramming, knowledge in developmental biology, unique new tools that we have built to define the molecular mechanisms of how a CM fate is established in a non-CM cell without transiting through the traditional developmental CM specification and differentiation route. Based on our preliminary data, we hypothesize that reprogramming factors Gata4, Mef2c and Tbx5 act in a stoichiometrically and temporally coordinated fashion upon a privileged subpopulation of CFs to overcome major epigenetic barrier(s) and induce CM fate. Successful completion of this proposal will define the molecular components and determine the optimal condition for iCM reprogramming. Our studies will provide novel insights into fundamental molecular mechanisms underlying cardiac cell fate acquisition and cell plasticity regulation.